Fluorescence spectrophotometry using multiple reflections to enhance sample absorption and fluorescence collection

ABSTRACT

Apparatus for enhancing the sensitivity of fluorescence spectrophotometry is described. Enhanced sensitivity is obtained by a novel sample cell construction in the form of a flat plate providing multiple exposure of the sample to the exciting radiation to increase absorption, and providing concentration of the fluorescent radiation to a small aperture for more efficient collection. The novel cell also provides easy separation of the exciting and fluorescent radiations.

United States Patent [191 Harrick 51 Feb. 6, 1973 54] FLUORESCENCESPECTROPHOTOMETRY USING MULTIPLE REFLECTIONS TO ENHANCE SAMPLEABSORPTION AND FLUORESCENCE COLLECTION [76] Inventor: Nicolas J. Harrick, Croton Dam Road, Ossining, NY. 10562 221 Filed: June7, 1971 21Appl.No.: 150,687

[52] US. Cl ..250/71 R, 250/77 [51] Int. Cl ..G0ln 21/16,

[58] Field of Search ..250/71, 77, 80, 71 R [56] References Cited UNITEDSTATES PATENTS 3,604,927 Hirchfield ..356/74 3,491,366 1/1970 Harrick..356/98 3,591,287 7/l97l Hannis ..356/51 3,470,261 9/1969 Roberts...260/67l 2,971,429 2/1961 Howerton ..250/71 R Primary Examiner-.lamesW. Lawrence Assistant Examiner-Harold A. Dixon Attorney-Jack Oisher [57]ABSTRACT Apparatus for enhancing the sensitivity of fluorescencespectrophotometry is described. Enhanced sensitivity is obtained by anovel sample cell construction in the form of a flat plate providingmultiple exposure of the sample to the exciting radiation to increaseabsorption, and providing concentration of the fluorescent radiation toa small aperture -for more efficient collection. The novel cell alsoprovides easy separation of the exciting and fluorescent radiations.

14 Claims, 12 Drawing Figures F'LUORESC ENCE PATENTEI] F E8 6 I973 SHEET20F 2 Fig. 4

Fig. 5

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' INVENTOR.

N. J. HARRICK hT'roRNEY EXCITATION FLUORESCENCE FLUORESCENCESPECTROPIIOTOMETRY USING MULTIPLE REFLECTIONS TO ENHANCE SAMPLEABSORPTION AND FLUORESCENCE COLLECTION This invention relatestofluorescence spectrophotometry, and in particular to a novel sample cellfor use therein to enhance sensitivity.

Fluorescence spectrophotometry apparatus comprises an excitation sourcegenerating a range of wavelengths for causing an unknown sample tofluoresce, an excitation monochromator to permit selection of thewavelength incident upon the sample housed in a suitable cell, anemission monochromator for selecting a particular wavelength of thespectral distribution of the fluorescent radiation emitted by thesample, and a photodetector, usually a photomultiplier tube, forconverting the received radiation into an electrical signal proportionalto the intensity of the received radiation. In the commercialinstruments, the excitation and fluorescent beams are oriented90 withrespect to each other in order to ensure adequate separation. Aftersuitable amplification, the electrical signal is usually displayed on anXY recorder or stripchart recorder. The resultant curves give thefluorescent intensity as a function of wavelength and may be used forqualitative and'quantitative analysis of the sample. Such apparatus arenoted for their high sensitivity, but modern science requires evenhigher sensitivity than that available from current instrumentsespecially for very small samples, known as microsamples.

Canadian Spectroscopy, 10, 128 (1965) suggests one technique to increasethe efficiency of excitation of film samples using multiple reflectionsof the exciting radiation in a thin transparent plate which supports thesample. The fluorescent radiation emitted from the sample is detected bya phototube located opposite the flat side of the plate and thus nofluorescence concentration results. Such a cell constructionis notusable with conventional double monochromator apparatus employing anemission monochromator because the fluorescent radiation extends overtoo large an area and therefore it is not readily focused for efficientuse in the emission monochromator.

In a subsequent publicationin Applied Optics, 6, 715 (1967), animprovement is described which attempts to combine multiple reflectionof the exciting'beam with collection of the emitted fluorescence byshaping the thin plate into a hemisphere, withthe sample on the curvedsurface and a photodetector ofa small diameter opposite the flatsurface, the exciting radiation entering the free annulus surroundingthe photodetector. The disadvantages of this construction are acomplicated cell shape which is difficult to fabricate and maintain byrepolishing, and insufficient collection of the emitted fluorescence, asubstantial portion of which is lost via the annulus. A more importantdisadvantage is inadequate separation of the exciting beam from theemitted fluorescence, since much of the incident excitation radiationwill also impinge upon the photodetector. As will be understood, theexcitation intensity is usually much stronger than the fluorescentemission.

The chief object of the invention is a novel cell construction offeringenhanced interaction of the exciting,

radiation with the sample, efficient collection of the emittedfluorescence, and substantially complete separation of theexcitation andfluorescent radiations.

A further object of the invention is a novel cell construction ofrelatively simple shape which is easy to fabricate, and well adapted foruse in a double monochromator fluorescence spectrophotometer.

These and other objects as will appear hereinafter are achieved, inaccordance with the invention, with a cell construction comprising aflat optically transparent plate having opposed polished major surfaces,preferably one reflecting short edge, preferably reflecting long edges,at least one flat polished major surface of the plate serving to receivea sample'to be analyzed, and means at the opposite short edge adapted toallow a substantial part of any fluorescent radiation present in theplate to exit from the plate at a different angle from any excitationradiation introduced into the plate whereby the two radiations can besubstantially separated from one another. In accordance with theinvention, the excitation radiation is caused to multiply reflect withinthe plate between the polished major surfaces increasing interactionwith the sample, and any fluorescent radiation emitted from the sampleand entering the plate is brought by multiple reflections to one shortedge' of the plate and thus concentrated to a small aperture and therecaused to exit toward the emission optics.

The invention will now be described in greater detail with reference tothe accompanying drawing, wherein FIG. '1 is a schematic view of aconventional double monochromator fluorescence spectrophotometer;

FIGS. 2 and 3 are plan and side views of one embodiment of my cellconstruction;

FIGS. 4, 5 and 6 are plan side and cross-sectional views of anotherembodiment of my invention;

FIG. 7 is a plan view of still another embodiment of my invention, withFIGS. 8 and 9 illustrating two modifications;

FIGS. 10 and 11 show two prism constructions for introducing theexcitation beam into my novel cell; and

FIG. 12 shows my novel cell substituted for the conventional cell of aspectrophotometer.

FIG. 1 illustrates a typical conventional double monochromatorfluorescence spectrophotometer. It

, comprises an excitation source 10 generating a range of wavelengths 11which are passed through an excitation monochromator l2, 13, 14 forselecting one or more wavelengths for exciting a sample intofluorescence.

The monochromator is shown schematically as a mirror I2, a grating 13and a mirror 14. The selected wavelengths are passed through an exitslit 15 usually forming a narrow vertical aperture and are focussed ontoa portion of a sample 17 contained within a sample cell 16. The sample17 is often a liquid to be analyzed. Fluorescence from the excitedsample portion which exits 18 from the cellat right angles to theexcitation radiation, is collected through an entrance slit 19 of theemission optics. The entrance slit 19 also usually forms a narrowvertical aperture. The fluorescent radiation is then passed through anemission monochromator 20, 21, 22 for analysis, comprising mirrors 20,22 and grating 21, and the selected wavelengths then impinge upon aphotodetector 23 which is usually a photomultiplier tube (PMT). As willbe appreciated from the geometry illustrated, the extent of interactionof the excitation beam with the sample is limited thus reducing theabsorption possibilities, the extent of collection of the fluorescentemissions is of the radiations, but none so far have been successful incombining increased interactions, increased collection'efficiency,reduction of the collected fluorescence to a small aperture, and goodseparation of the exciting and fluorescent radiations. In my invention Iuse as the sample cell to be substituted for the prior art cell 16 aflat plate of optically transparent material. Such a plate isillustrated in a top view in FIG. 2 and side view in' FIG. 3. The plate,designated-25, has major flat opposed parallel surfaces 26 and 27, bothof which are polished to provide loss-less total reflection and one orboth of which may receive a film sample 30 to be analyzed. One shortedge surface'31 of the plate has metallization 32 to form a reflectingsurface or mirror. The two long edge surfaces33 and 34 also havemetallizations 35' thereon to form reflecting surfaces or mirrors. Theopposite shortedge surface 36 has no metallization and is polished flatandremains transparent a'ndservesas a window to allow radiation toenter-and exit fromthe plate 25. All edge surfaces are perpendicular tothe'r najor surfaces 26,27. Contacting the transparent short edge 36 atits flat side is a half round optically transparent 1 member 40, thatis, a hemicylinder. The half round 40 serves to introduce and extractradiation from the plate 40. It will also be appreciated that the halfround 40 contacting the short edge can i be replaced by its opticalequivalent two quarter rounds, one sitting on surface 26 adjacent theshort edge 36, and the other sitting on surface 27 ad.-

jacent the same short edge, with their curved surfaces facing outward.

' Assume that exciting radiation 39 is introduced into the half round40?- at a 45 angle to theplane of the plate, andassume further that thecritical angle c of tions. Thus all radiation at angles between Ocand 90becomes trapped within the plate by the major surfaces 25, 27. Howeverthe radiation incident on the transparent edge 36 at angles between 0and 00 (which equals between 90 and 90-6c relative to the majorsurfaces) will be emitted from the transparent edge. The radiationincident on the transparent short edge at angles relative to the majorsurfaces between 00 and 90-0c, in the absence of the .half round 40,remains permanently trapped within the plate and becomes reabsorbed.Also, in the absence of the half round 40, the radiation emitted fromthe transparent short edge fans out to 6 i 90. 7

The half round 40 is used to extract all of the energy trapped by themajor surfaces within the plate, and thus all the energy incident on theedge surface 36 at angles relative to the major surfaces 26, 27 between00 and 90, which relative to the edge 36 becomes 90 0c and 90, shown inFIG. 2 by the dashed lines designated 42.

As the emerging radiation suffers no refraction in its short edgesurface 31 ensures that all the'energy trapped by the major surfaces isextracted through the transparent edge 36.

As will be observed, the exciting radiation has, due to I multiplereflections through theplate, increased opportheplate is below 45, Underthese conditions, the exciting radiation will propagate by multipletotal reflections down the length of the plate to the, right, reflectfrom theimirrored short edge 3l,-and propagate to the .left up theplate'and exit from the short edge 36into'the half round 40 still at 45butopposite to ray 39. On the bottom surface of the element 40 islocated a strip 38 of metallization serving as a. mirror. The excitationwilltion will enter the plate 25. Of the reentering radiation,

that fraction which is incident on the major surfaces 26, 27 at an angleexceeding 0cwill undergo total reflection and propagate through theplate by multiple reflec-.

tunity to interact with the 1 sample and cause fluorescence, thusproducing enhanced absorption. That fraction of the fluorescenceentering the plate and trapped by total reflection by the major surfacesis efficiently collected within the plate and brought to the smallaperture represented by the transparent short edge 36. By means ofthehalf round 40, most of the trapped energy can be extracted" anddirected toward the entrance slit 19 of the emission optics. Asillustrated inFIG. 2, the collected radiation can extend between thelines 43, 44 and thus be adequately separated from the excitingradiation 39.-'Tlie structure illustrated in FIGS. 2 and 3 offers thefurther advantage that the angle of incidence for-the excitation beamcan be variedcontinuously thereby changing the depth'of. penetration ofthe exciting beam into the sample.

The cell structure of FIGS. 2 and 3 is especially useful for analyzingthis film liquid samples, which can be placed in contact with one orboth of the polished major surfaces 26, 27. The film sample can have arefractive index n the same as, lower, or higher than that of the plate25. Any optically transparent material can be used for the plate 25, butone with a low refractive index n is preferred, because 60 will belower. Two

preferred materials are quartz (n 1.5, 0c 42) and radiation at a platelocation different from the short edge surface from whence thefluorescence emerges and thus further increase their separation. Forexample, the exciting radiation can be introduced at one of the longedges, though this may also increase loss of the fluorescent energy. Butthe introduction of the excitation at a long edge offers the possibilityof other structures for extracting the trapped fluorescence from theshort edge and thus through a small aperture.

One such structure in accordance with the invention is a light funnel,which is illustrated in a top view in FIG. 4, a side view in FIG. 5, anda cross-sectional view in FIG. 6. The same reference numerals are usedfor corresponding elements. In this embodiment, one long edge surfaceand one short edge surface is metallized at 35 and 32. A part 45 of theopposite long edge surface is bevelled flat at a 45 angle. This angle isnot critical and other angles can be used. The remainder of that longedge surface 46 is polished flat and metallized 47 similarly to theopposite long edge. The bevel 45 is used as a window to introduce theexciting radiation 41 at an angle to the major surfaces exceeding thecritical angle to cause multiple reflections therefrom and also slightlyoblique to the vertical to cause the beam to zig-zag across the width ofthe plate and thus propagate down the length of the plate 25 as shown.The emitted fluorescent radiation that enters and becomes trapped withinthe plate propagates along its length. The extraction means therefor isa funnel 50 with polished surfaces which can be optically contacted tothe short edge of the plate as shown or be fabricated as an integralpart of the plate. The fluorescent radiation crosses from the plate 25into the funnel 50 through the short edge and as it propagates down thefunnel towards the thinner-edge multiply reflects from its surfaces atincreasing angles of incidence due to the tapering funnel. As criticalangle is approached, the radiation emerges from the funnel at neargrazing incidence, shown at 51, in the forward direction (towards thetip) and to the left in FIGS. 4 and 5, and thus can be directed into theemission optics.

Another construction for extracting the trapped fluorescent energy is asimple bevel 55, which is illustrated in FIG. 7, at the shorttransparent edge. Here, however, instead of changing the angle ofincidence gradually, as in the funnel extractor of FIGS. 4 and 5,.

the angle changes in one jump by one reflection from the bevel surface.However, by.a suitable choice of bevel angle, which also depends uponthe refractive index of the plate, a substantial part of the trappedenergy can be extracted so as to issue in the forward direction, i.e.,away from the plate. The manner of choosing a suitable bevel angle willbecome apparent to those skilled in this art from a consideration of thecases of bevel angles of 60 and 45, illustrated in FIGS. 8 and 9,respectively.

FIG. 8 illustrates the case for a 60 bevel. The various solid linearrows show the different directions that can be taken by the emergingfluorescent radiation. Some of the radiation will be emitted from thebevel largely in the forward direction of its tip. The remainder will betotally reflected from the bevel surface and emerge ing in the backwarddirection will be lost. FIG. 9 illustrates the situation under the sameconditions for a 45 bevel. In this case, part emerges as before from thebevel surface in the forward direction, but the part emitted from thelower major surface is generally in the backward direction and notusable. Thus the larger bevel angle is preferred. A suitable bevel anglerange for most practical purposes is about 50- With the 60 bevel forexample, at least 50 percent of the emitted radiation is in the forwarddirection. In general, the higher the refractive index of the plate andthe smaller the bevel angle, the more energy will be emitted by themajor surface 27. The light funnel of FIG. 4 may be regarded as a bevelwith a very small angle, and thus nearly all of the energy is emittedfrom the major surfaces.

In the case of the FIG. 8 embodiment employing the bevel 55 as theextraction means, the exciting radiation can be introduced transverselyat a bevelled portion along a long edge, as illustrated at 45 in FIG. 5,or it can be introduced into the extraction bevel as shown by the dashedarrow 61 in FIG. 8. Another way of introducing transversely the excitingradiation is by way of simple right angle prisms mounted on a majorsurface close to a long edge, in a position approximately correspondingto that of the bevel 45 of FIG. 5. This is illustrated incross-sectional views in FIGS. 10 and 11, showing prisms 65, 66 arrangedin two ways for introducing the excitation beam. When the excitationradiation is transverse to the fluorescent radiation, Raman Spectroscopycan be accomplished, since the latter requires excitation andobservation at right angles.

As mentioned above, the half round extractor 40 can be used with thinfilm samples whose refractive index has any value relative to that ofthe plate. With the embodiments of FIGS. 4-9, the rule is the same forthin film samples.

My novel cell construction is also useful for analyzing liquids andsolids, for example, immersing the cell plate in a liquid sample.However, in this case, critical reflection of the fluorescent radiationentering the plate cannot be obtained. However, that fluorescentradiation which has a direction toward the extraction short edge with anangle of incidence relative thereto between 6c and will be collected.The cell can be viewed as an extended window immersed in the liquid orsolid sample and collecting fluorescence over 'a much deeper depth thanwould ordinarily be possible with a conventional cell, since thefluorescence emerging via the cell plate avoids being reabsorbed by thesample. In this case, the plates refractive index should be higher thanbut still close to that of thesample, since only the fluorescentradiation between 0 and 0c relative to the major surfaces can enter theplate. If the entire plate is immersed, the half round extractor of FIG.2 is preferred as it permits also the exciting radiation to beintroduced via the short edge.

One of the features of the invention is that my novel cell constructioncan be substituted for the conventional cell in known doublemonochromator fluorescence spectrophotometers with no change in theoptics or at most with very little change. FIG. 12 shows this. Thespectrophotometer geometry is the same as in FIG. 1, only the slits 15and 19 being shown for simplicity. The cell 25 of FIG. '7 is positionedas in FIG. 8

such that the excitation 61 is normal to the bevel edge 55, and thefluorescence directed alongv the path 18 from the plate 1 which passesthrough the slit 19 is analyzed by the emission monochromator aspreviously described.

While I have described my invention in connection with specificembodiments and applications, other modifications thereof will bereadily apparent to those skilled in this art without departing from thespirit and scope of the invention as defined in the appended claims. 7

What is claim is:

l. A fluorescence spectrophotometer comprising a sample plate ofoptically transparent material having major opposed planar parallelsurfaces defining at least a first edge surface for admitting ortransmitting an optical beam and at least a second edge surface remotefrom the first edge surface, both major surfaces being polished with atleast one adapted to receive a sample to be analyzed, means fordirecting an excitation radiation beam into the plate at an angleexceeding the critical angle whereby the, excitation beam propagatesthrough the plate by multiple reflections from at least the majorsurfaces, said excitation beam establishing at said one major surface ateach reflection an evanescent wave capable of interacting with thesample and causing it to emit characteristic fluorescent radiation, someof which enters the plate and impinges on a surface at an angleexceeding the critical angle causing the fluorescent radiation topropagate through the plate by multiple reflections, and means locatedadjacent said first edge surface at such a position as to receive anddetect substantially only that fluorescent radiation which propagatesthrough the plate by multiple reflections and exits from the plate viasaid first edge surface, whereby both sample excitation andemission-collection are enhanced by multiple internal reflections in theplate.

2. A fluorescence spectrophotometer as set forth in claim 1 and furthercomprising a source of excitation radiation, and an excitationmonochromator for receiving the excitation radiation and passingselected wavelengths thereof to the excitation beam directing means,said fluorescent radiation detecting means comprising an emissionmonochromator for receiving the exiting fluorescent radiation andpassing selected wavelengths thereof to a photodetector.

'3. A fluorescence spectrophotometer as set forth in claim 2 whereinsaid sample plate has reflecting surfaces at all of its edge surfaceswith the exception of the first edge surface, and the excitationradiation directing means is located adjacent the first edge surface forreceiving and exiting aperture, and the excitation and emissionmonochromators each have slit systems whose size generally correspondsto that of the given aperture. 4 i

5. A fluorescence spectrophotometer as set forth in claim 1 wherein thefluorescence receiving means comprises a hemicylindrical structurelocated at the first edge surface.

. A fluorescence spectrophotometer as set forth ll'l claim 1 wherein thefluorescence receiving means comprises a light funnel whose large edgeis located adjacent the first edge surface.

7. A fluorescence spectrophotometer as set forth in claim 1 wherein thefirst edge surface is beveled flat at an angle relative to the majorsurfaces.

8. A sample cell for use in fluorescence spectrophotometry comprising aflat plate of optically transparent material having major opposed planarparallel polished surfaces defining opposed short edge surfaces andopposed long edge surfaces, at least one of said long edge surfaces andone of said short edge surfaces being perpendicular to the majorsurfaces and being beam reflectors, at least one of said major surfacesserving to receive a sample to be analyzed, said other short edgesurface being optically transparent for receiving and transmittingoptical radiation, and means associated with said other short edgesurface to permit fluorescent radiation trapped in the plate by multiplereflection by the major surfaces to exit from said other short edgesurface at a different angle from that of excitation radiationintroduced into the plate.

9. A sample cell as set forth in claim 8 wherein the reflecting surfacesformed on the one short edge and the one long edge surfaces are formedby metallizations thereon.

10. A sample cell as set forth in claim 9 wherein said other short edgesurface is beveled flat at an angle between about 50 and relative to themajor surfaces so as to permit a large fraction of fluorescent radiationin the plate to exit therefrom in a forward direction.

11. A sample cell as set forth in claim 9 wherein said other short edgesurface extends perpendicular to the major surfaces, and ahemicylindrically shaped optically transparent member is locatedadjacent said other short edge surface.

12. A sample cell as set forth in claim 11 wherein a side portion of thehemicylindrically shaped member is metallized to reflect excitationradiation.

13. A sample cell as set forth in claim 9 wherein a light funnel iscontacted via its long end to saidother short edge surface.

14. A sample cell as set forth in claim 8 and including means locatedadjacent part of a long edge surface for introducing excitationradiation into the plate substantially transverse to its longitudinaldirection and at an angle relative to the major surfaces which exceedsthe critical angle.

1. A fluorescence spectrophotometer comprising a sample plate ofoptically transparent material having major opposed planar parallelsurfaces defining at least a first edge surface for admitting ortransmitting an optical beam and at least a second edge surface remotefrom the first edge surface, both major surfaces being polished with atleast one adapted to receive a sample to be analyzed, means fordirecting an excitation radiation beam into the plate at an angleexceeding the critical angle whereby the excitation beam propagatesthrough the plate by multiple reflections from at least the majorsurfaces, said excitation beam establishing at said one major surface ateach reflection an evanescent wave capable of interacting with thesample and causing it to emit characteristic fluorescent radiation, someof which enters the plate and impinges on a surface at an angleexceeding the critical angle causing the fluorescent radiation topropagate through the plate by multiple reflections, and means locatedadjacent said first edge surface at such a position as to receive anddetect substantially only that fluorescent radiation which propagatesthrough the plate by multiple reflections and exits from the plate viasaid first edge surface, whereby both sample excitation and emissioncollection are enhanced by multiple internal reflections in theplate.
 1. A fluorescence spectrophotometer comprising a sample plate ofoptically transparent material having major opposed planar parallelsurfaces defining at least a first edge surface for admitting ortransmitting an optical beam and at least a second edge surface remotefrom the first edge surface, both major surfaces being polished with atleast one adapted to receive a sample to be analyzed, means fordirecting an excitation radiation beam into the plate at an angleexceeding the critical angle whereby the excitation beam propagatesthrough the plate by multiple reflections from at least the majorsurfaces, said excitation beam establishing at said one major surface ateach reflection an evanescent wave capable of interacting with thesample and causing it to emit characteristic fluorescent radiation, someof which enters the plate and impinges on a surface at an angleexceeding the critical angle causing the fluorescent radiation topropagate through the plate by multiple reflections, and means locatedadjacent said first edge surface at such a position as to receive anddetect substantially only that fluorescent radiation which propagatesthrough the plate by multiple reflections and exits from the plate viasaid first edge surface, whereby both sample excitation and emissioncollection are enhanced by multiple internal reflections in the plate.2. A fluorescence specTrophotometer as set forth in claim 1 and furthercomprising a source of excitation radiation, and an excitationmonochromator for receiving the excitation radiation and passingselected wavelengths thereof to the excitation beam directing means,said fluorescent radiation detecting means comprising an emissionmonochromator for receiving the exiting fluorescent radiation andpassing selected wavelengths thereof to a photodetector.
 3. Afluorescence spectrophotometer as set forth in claim 2 wherein saidsample plate has reflecting surfaces at all of its edge surfaces withthe exception of the first edge surface, and the excitation radiationdirecting means is located adjacent the first edge surface for directingthe beam through the first edge surface, said excitation beam directionbeing different from that of said fluorescent radiation exiting from theplate via said first edge surface.
 4. A fluorescence spectrophotometeras set forth in claim 2 wherein the first edge surface has a given beamreceiving and exiting aperture, and the excitation and emissionmonochromators each have slit systems whose size generally correspondsto that of the given aperture.
 5. A fluorescence spectrophotometer asset forth in claim 1 wherein the fluorescence receiving means comprisesa hemicylindrical structure located at the first edge surface.
 6. Afluorescence spectrophotometer as set forth in claim 1 wherein thefluorescence receiving means comprises a light funnel whose large edgeis located adjacent the first edge surface.
 7. A fluorescencespectrophotometer as set forth in claim 1 wherein the first edge surfaceis beveled flat at an angle relative to the major surfaces.
 8. A samplecell for use in fluorescence spectrophotometry comprising a flat plateof optically transparent material having major opposed planar parallelpolished surfaces defining opposed short edge surfaces and opposed longedge surfaces, at least one of said long edge surfaces and one of saidshort edge surfaces being perpendicular to the major surfaces and beingbeam reflectors, at least one of said major surfaces serving to receivea sample to be analyzed, said other short edge surface being opticallytransparent for receiving and transmitting optical radiation, and meansassociated with said other short edge surface to permit fluorescentradiation trapped in the plate by multiple reflection by the majorsurfaces to exit from said other short edge surface at a different anglefrom that of excitation radiation introduced into the plate.
 9. A samplecell as set forth in claim 8 wherein the reflecting surfaces formed onthe one short edge and the one long edge surfaces are formed bymetallizations thereon.
 10. A sample cell as set forth in claim 9wherein said other short edge surface is beveled flat at an anglebetween about 50* and 70* relative to the major surfaces so as to permita large fraction of fluorescent radiation in the plate to exit therefromin a forward direction.
 11. A sample cell as set forth in claim 9wherein said other short edge surface extends perpendicular to the majorsurfaces, and a hemicylindrically shaped optically transparent member islocated adjacent said other short edge surface.
 12. A sample cell as setforth in claim 11 wherein a side portion of the hemicylindrically shapedmember is metallized to reflect excitation radiation.
 13. A sample cellas set forth in claim 9 wherein a light funnel is contacted via its longend to said other short edge surface.